The wearing of soft contact lenses is a known risk factor for the development of a progressive keratitis caused by free-living amoebae of the genus Acanthamoeba. These common environmental protozoa are highly resistant to environmental degradation and are also resistant to most commonly used antibiotics. Species identification of Acanthamoeba remains problematic, although proper characterization of species is necessary for epidemiologic and perhaps therapeutic purposes. Variability seen with in vitro susceptibility testing data are also confusing, and may be the result of differing test methodologies or of innate strain differences. Initial studies in this project will use established morphologic and biologic criteria to characterize both clinical and environmental isolates of Acanthamoeba. While currently maintaining a number of isolates recovered from ocular infections, other strains will be isolated from natural substrates and compared for biological and physiological differences. Susceptibility testing will subsequently be performed on all isolates using in vitro microtiter broth dilution techniques, and both inhibitory and cidal endpoints to a broad range of anti-protozoals, anti-fungals, and other drugs will be determined. Test methodologies will be standardized for both culture conditions and endpoint criteria to minimize variability of results. Preliminary data from these studies will be used in future projects to design immunologic and genetic methods for the rapid detection of Acanthamoeba from ocular and other specimens. The development of a standardized in vitro drug screening test will aid in the future identification of metabolic pathways which may be targeted directly by specific inhibitors or analogs.